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COLTURE CELLULARI
Colture primarie
Maria Chiara Zatelli
Sezione di EndocrinologiaUniversità di Ferrara
Diretore: Prof. Ettore degli Uberti
Colture cellulariDEFINIZIONEProcedimento complesso mediante il quale le cellule sonocoltivate in condizioni controllate, al di fuori del loroambiente naturale.
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Colture primarie
cellule animalipiantemiceti
organismi multicellulari
virusbatteri protozoi
organismi unicellulari
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Colture primarie
Purificazione da sangue�Leucociti
� proliferazione in vitro�
� Eritrociti
Digestione enzimatica di tessuti molli�Cellule nucleate
Coltura di espianto� Frammenti di tessuto in terreno di coltura
Isolamento delle cellule
Colture cellulari
� Colture primarie
� Linee stabilizzate
� Linee trasformate
� Linee ingegnerizzate
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Colture primarie
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Colture primarie
Colture cellulari derivanti da un singolo individuo � durata limitata� sviluppo di senescenza – interruzione della proliferazione
Colture primarie
Le linee cellulari immortalizzate hanno acquisito la capacità di proliferare in modo indefinito grazie a mutazioni casuali o modifiche apposite
Linee stabilizzate
Colture primarie
� Organo e/o tessuto disgregato meccanicamente o enzimaticamente in cellule singole o clumps
� Coltura in sospensione e/o su substrato solido
� Capacita’ proliferativa limitata
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Perché si coltivano?
� Per espandere i cloni cellulari da indagare
� Per investigare le caratteristiche biochimichee fisiologiche delle cellule (endocrine)
� Per capire le basi molecolari e cellulari dellepatologie d’organo
� Per valutare l’efficacia di agenti terapeutici
� Per testare e sviluppare nuovi materiali
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Colture primarie
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Colture primarie
Cosa serve? Incubatore per colture cellulari
Temperatura = 37°CAtmosfera controllata = 5% CO2 e 95% aria per le
cellule di mammifero
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Colture primarie
Cosa serve? Cappa a flusso laminare “biohazard”
flusso continuogaranzia di sterilità all’interno della cappasterilizzazione mediante UV
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Colture primarie
Cosa serve? Microscopio ottico
visione invertitacontrasto di fasepossibilità di fotografare
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Colture primarie
Cosa serve? Terreno di colturaA seconda delle caratteristiche del tessuto è possibilepreparare (o acquistare) uno specifico terreno di coltura,Le variabili più importanti sono
pHconcentrazione di glucosiopresenza di fattori di crescitaconcentrazione del siero
(solitamente siero fetale bovino)altri nutrienti (aminoacidi, vitamine)
Potenzialecontaminazione
con virus o prioni
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Colture primarie
Come si fa? • Prelevare il tessuto fresco in condizioni di sterilità • Trasferire il tessuto fresco in una petri
• Lavare con PBS
• Dissezionare eliminando grasso
e tessuto necrotico
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Colture primarie
Come si fa?
• Lavare ripetutamente
• Rimuovere il terreno in eccesso
e ricoprire con terreno di coltura
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Colture primarie
Come si fa? • Trasferire i pezzi in una fiasca da 25 cm2
• Aggiungere enzimi e porre in orbitalincubator a 37° per 1-2 ore
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� Tripsina : (idrolasi) massima attività a 37°, induce una maggiore disgregazione ma può danneggiare le cellule.Per minimizzare il danno è possibile usare tripsinaa 4°C per permettere all’enzima di penetrare neltessuto con poca attività
� Collagenasi : (metallo-proteinasi) determina una minore disgregazione ma è meno aggressiva
� Ogni tessuto richiede condizioni diverse
Enzimi
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Come si fa? • Passaggi in aghi di diametro sempre minore
(18 – 22 g)
Si ottiene una sospensione cellulare mista
� Centrifugare ed eliminare
il surnatante
� Risospendere il pellet in 1-5 ml di RPMI
� Pelevare 10 ul della sospensione cellulare e contare in camera di Burker
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o E’ caratterizzata da una griglia standardizzata
costituita da 9 quadrati.
o La media del numero di cellule che si contano nei 4
quadrati viene moltiplicata per un valore fisso 104 (poichè
ogni quadrato rappresenta un volume totale di 0,1 mm3) e
si ottiene quindi il numero di cellule in 1 ml di soluzione
Camera di burker
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• Osservare al microscopio ottico le cellule galleggianti nel mezzo di coltura, in modo da valutare la congruità delle cellule piastrate• Seminare nelle piastre da 96 well 20.000cells/wells• Porre nell’incubatore termostatato a 37°Ce 5% CO
2, dopo circa 2-3 ore è possibile
osservare le cellule che iniziano adattaccarsi alla piastra ed acquisire lapropria forma
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Come si fa?
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Come si fa?
Crescita cellulare in 3-DPiù simile al tessuto in vivo
Tecnicamente molto complessa
Densità di semina (numero di cellule/ml di terreno di coltura) � Influenza le caratteristiche e la differenziazione cellulare
Coltura in sospensione vs. in adesione
alta densità Superficie di crescita:plasticamicrocarriers coating
colture organitipiche
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Colture primarie
Come si fa? sospensione/monostrato
Le cellule continuano a crescere fino a riempire tutto lo spazio disponibile
Deplezione di nutrientiAccumulo di cellule
apoptotiche/mecrotiche Inibizione da contatto
Induzione della differenziazione
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Come si fa?
Tutte le operazioni (cambio del mezzo do cuùoltura, passaggio delle cellule, trasfezioni)
devono essere svolte in CONDIZIONI DI STERILITA’
sospensione/monostrato
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Colture primarie
Come si fa? Cappa a flusso laminare
AntibioticiAntimicotici
Indicatori di variazione del pH
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Sostituzione del mezzo di coltura
PassaggioTrasferimento di un piccolo numero di cellule in una nuova fiascaSe fatto regolarmente evita la senescenza
Semina in piastra
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E ora ?
Incubazione
Raccolta del mezzo di coltura
Dosaggi(ormonali)
Saggi diVitalità
ApoptosiAttivazione vie
del segnale
Immunofluorescenza
Fissazione su vetrino
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Native MCF7
rMCF7
DoxoControl NS-398
A
B
C
D
NS-398 + Doxo
E
F
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Produzione di vacciniProduzione biotecnologica con tecnica del DNA ricombinante di
enzimiormonianticorpi monoclonaliinterlauchinelinfochineagenti antineoplastici
�Soprattutto per proteine glicosilate e/o dotate di modifiche post-traduzionali che non possono essere riprodotte in ambiente batterico
ERITROPOIETINA
L’alternativa alle colture cellulari di mammifero sono costituite da cellule di insetto, cellule vegetali, cellule embrionali
Applicazioni
Pancreatic endocrine tumors
Does targeting Protein Kinase restrain proliferation in human pancreatic endocrine tumors?
PKC
βII
δ
PKC: key enzymes of cellular dynamics
APOPTOSIS
ANGIOGENESIS
SURVIVAL
PROLIFERATION
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Pancreatic endocrine tumors
20
40
60
80
100
120
140
160
180
% v
s. c
ontr
ol
cell viability
CgA secretion
Insulin secretion
* *
*
*
* *
Enzastaurin (µM) 0 1 5 10 0 1 5 10
IGF-I (nM) 0 0 0 0 100 100 100 100
ENZASTAURIN
PKCβII inhibitor
Riduces neoplastic
cell division
Inhibits tumor
vascularizationEnhances apoptosis
Reduces PNN
primary culture cell
viability and CgA
secretion
Molè et al. submitted to Endocr-Relat Can
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Pancreatic endocrine tumors
ENZASTAURIN
BON-1 cell line
� cell viability
� DNA synthesis
� apoptosis
� GSK3β phosphorylation
� CgA secretion
antiproliferative effects
antisecretory effects
Molè et al. submitted to Endocr-Relat Can
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Pancreatic endocrine tumors
0 5 µM
6 h
12 h
24 h
ENZASTAURIN
BON-1 cell line
� CgA expression
Molè et al. submitted to Endocr-Relat Can EFE 2012
Colture primarie
Pancreatic endocrine tumors
Merged
0 5 µM
10 µm
BON1
0 5 µM
10 µm
PNN
PKC-βII
PKC-δ
ENZASTAURIN
BON-1 cell line
isoform delocalization
Molè et al. submitted to Endocr-Relat Can EFE 2012
Colture primarie
Pancreatic endocrine tumors
Enzastaurin (µM) 0 5 0 5
IGF-I (nM) 0 0 100 100
PKC-βII
PKC-δ
β actin
76,8 kDa
77,4 kDa
43 kDa
ENZASTAURIN
BON-1 cell line
no protein changes
Molè et al. submitted to Endocr-Relat Can
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Colture primarie
Pancreatic endocrine tumors
ENZASTAURIN
PKC may represent a new pharmacological target
for pancreatic endocrine tumors medical therapy
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Pancreatic endocrine tumors
72 primary PETs7 matched metastases10 normal pancreatic samples
validation by QPCR or IHC
possible molecular signatures? Missiaglia et al 2010 J Clin Oncol 28:245-55
Absent or low SSTR2 in insulinomas vs. nonfunctioning tumors
FGF13 expression � liver metastases � disease-free survival
� disease-free and overall survival
� TSC2 and PTEN
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Sandoval et al. 2009 Nat Rev Drug Disc 8: 386-98
TSC2mTOR up-stream signalling pathway
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Astrinidis et al. 2005 Oncogene 24: 7475–7481
TSC2 physiologically inhibits mTOR activation
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large serine-threonine kinase
nutrients utilization
viability
-+
biological switch sensing changes in the cellular environment and helping cells respond EFE 2012
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TS
C2
mT
OR
AK
T
+--+
Normal neuroendocrine cell
TS
C2
mT
OR
AK
T
+--+
TSC2 mutated neuroendocrine cell
EF
E 2
01
2
Colture prim
arie
High p-mTOR expression in poorly differentiated
neuroendocrine carcinomas
potential therapeutic role for mTOR inhibitors
in neuroendocrine tumors
Clinical trials
RAD-001 inhibits neuroendocrine cell
proliferation
Grozinsky-Glasberg et al 2008 Neuroendocrinology 87:168Zitzmann et al. 2007 Neuroendocrinology 85:54-60
Shimizu et al 2010 Cancer Chemother Pharmacol 65:889-93
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RAD-001 In Advanced Neuroendocrine Tumors (RADIANT)
RADIANT-1: Phase 2 open label study of RAD-001 in advanced pancreatic neuroendocrine tumors after failure of chemotherapy
Stratum 1 115 patients
Stratum 245 patients
RAD001 10 mg/d + octreotide LAR 30 mg/4w
RAD001 10 mg/d
Yao et al. 2010 J Clin Oncol 28: 69-76
clinical benefit
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� VEGF secretion
immunosuppressant agent
� organ transplants rejection
antineoplastic activity
derivative of Rapamycin
mTOR inhibitor
mTORC1 protein
cell cycle G1-S phase
X
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Inoperable bronchial carcinoids are still orphan of medical therapy
BRONCHIAL CARCINOIDS
What about other endocrine tumors?
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Biomolecular analysis Pathology
Multidisciplinary group
surgery
Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies - University of FerraraDepartment of Medical and Surgical Sciences, University of PadovaDepartment of Thoracic Surgery, University of PadovaDepartment of Diagnostic Medical Sciences and Special Therapies, University of PadovaInstitute of Clinical Surgery, University of Ferrara
24 bronchial carcinoids
Zatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
Pati
ents
char
acte
rist
ics
14 ♂, 10 ♀51.5 ± 3.1 y
19 typical carcinoids5 atypical carcinoids
responders non responders
Cell viability response to
RAD001
primary cultures
Patients Age Sex Diagnosis TNM
1 49 F Typical T2N2
2 36 F Atypical T2N1
3 49 F Typical T3N0
4 29 M Typical T2N0
5 45 M Typical T2N0
6 52 M Typical T1N0
7 36 F Typical T2N1
8 74 F Typical T1N0
9 68 M Atypical T2N0
10 39 M Atypical T3N0
11 47 F Typical T2N0
12 76 M Typical T1N1
13 42 M Typical T2N0
14 30 M Typical T2N0
15 72 F Typical T1N0
16 72 M Typical T1N1
17 39 F Typical T2N0
18 72 M Typical T1N1
19 68 M Typical T2N0
20 41 M Atypical T3N0
21 50 F Typical T1N0
22 57 M Typical T2N1
23 45 M Typical T1N0
24 49 F Atypical T2N0EFE 2012
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N R
p-mTOR
mTOR
beta-actin
cell
viab
ility
(%
vs.
con
trol
)
All
Responder (15)
Non responder (9)
0
20
40
60
80
100
120
140
0 1 nM 10 nM 100 nM 1 µM
* **
* *
Everolimus
RAD001 reduces cell viability in BC with higher total and phosphorylated mTOR
Zatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
BC characteristics according to response to everolimus
Responders Non respondersp
(responders vs. non responders)
Age 53 ± 5.4 48 ± 4.7 n.s.
Gender (M/F) 8/7 5/4 n.s.
Smoking history (yes/no) 6/9 4/5 n.s.
Diameter (cm) 3.6 ± 0.4 2.16 ± 0.3 < 0.05
Lymphnode metastases (%) 10.4 3.1 < 0.02
Typical /atypical 10/5 9/0 < 0.05
Mitotic figures/mm2 1.7 ± 0.2 0.8 ± 0.1 < 0.01
CD105 (counts/mm2) 43.3 ± 9 25.3 ± 4.3 < 0.05
Plasma CgA levels (ng/ml) 496.8 ± 144 57.6 ± 2.1 < 0.05
Plasma PP levels (ng/L) 116.6 ± 21 51.2 ± 7.6 < 0.05
mTOR mRNA expression (fold vs. non responders)
900 1 < 0.01
Zatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
Cell viability
24 BC
15 RAD-responders
tumor diameter% local metastases
mitosesangiogenetic marker
CgA and PP plasma levelsmTOR expression
BC responding to everolimus are more aggressive
Zatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
0
10
20
30
40
50
mR
NA
cop
ies
x 1
04/µ
g to
tal R
NA
SSTR1 SSTR2 SSTR3 SSTR4 SSTR5
0
20
40
60
80
100
120
0 1 nM 10 nM 100 nM 1 µM
cell
viab
ility
(%
vs.
con
trol
)
w/o SOM230
with SOM230
* ** *
** *
*
Everolimus
SSTR2
SSTR3
SSTR5
SOM230
SOM230 reduces cell viability but
has no cooperative effectZatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
differentiation
w/o SOM230
with SOM230
0
20
40
60
80
100
120
0 1 nM 10 nM 100 nM 1 µM
CgA
sec
reti
on (
% v
s co
ntro
l)
* *** * *
*
Everolimus
Everolimus and SOM230 reduce CgA secretionwithout
cooperative effects
-
Zatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
Gong et al. 2007 Endocrinology 148:4489
but not only differentiation.. !!
+
+
Neu
roen
doc
rine
cel
l
+
AKT
mTOR
+
+
+
AKT
mTOR
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Neu
roen
doc
rine
cel
l
+
+
+
AKT
mTOR
+
+
+
+
AKT
mTOR
lower CgA secretionmight result in reduced
cell growth
Gong et al. 2007 Endocrinology 148:4489 EFE 2012
Colture primarie
angiogenesis
-
w/o SOM230
with SOM230
0
20
40
60
80
100
120
0 1 nM 10 nM 100 nM 1 µM
VE
GF
sec
reti
on (
% v
s. c
ontr
ol)
* ** * * * *
Everolimus
Everolimus and SOM230 reduce VEGF secretionwithout
cooperative effectsZatelli et al. Endocr Relat Cancer. 2010;17:719-29 EFE 2012
Colture primarie
Paez-Ribes et al. 2009 Cancer Cell 15: 220–231
Increased invasive phenotype after anti-VEGFR2 therapy
Increased tumor invasion after tumor-specific VEGF-A gene deletion
“…potent angiogenesis inhibition can alter the natural history of tumors by increasing invasion and metastasis …”
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angiogenesis
-
Chacko et al. 2009 J Clin Neurosci 16: 660–665
Angiogenesis is enhanced in aggressive pituitary adenomas
mTOR inhibitorsin invasive pituitary
adenomas??
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Everolimus dose-dependently reduces NFA cell viability in vitro
Cell viability
Zatelli et al. 2010 J Clin Endocrinol Metab 95(2):968-76
*p< 0.05**p< 0.01 vs. control
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Cell viability
Zatelli et al. 2010 J Clin Endocrinol Metab 95(2):968-76
40 NFA
12 RAD-non responders28 RAD-responders
median age (yr) 57 69
M/F 0.65 1.4
invasive 20 (71%) 7 (58%)
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Zatelli et al. 2010 J Clin Endocrinol Metab 95(2):968-76
RAD-responders
70% of the examined tumors
deriving mostly from younger female
patients with
invasive macroadenomas
patients likely not cured by surgery could be eligible for medical therapy with
mTOR inhibitors
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+
+
AKT
mTOR
+ -
p70S6K
+
-• reduces NFA cell viability
• promotes apoptosis
• reduces p70S6k activity
blocking IGF-I stimulatory effects
Zatelli et al. 2010 J Clin Endocrinol Metab 95:968
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Zatelli et al. 2010 J Clin Endocrinol Metab 95(2):968-76
Any synergism with DA and SSTR agonists?
Cabergoline significantly reduces NFA
cell viability
but
no additive effects with RAD001
SOM230 significantly reduces cell viability in SSTR5- NFA with additive effects with RAD001
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+
+
AKT
mTOR
+
p70S6K
+
Zatelli et al. 2010 J Clin Endocrinol Metab 95:968
-• blocks IGF-I induced VEGF secretion
• does not enhance the antisecretory
effects of DR and SSTR agonists
Cabergoline-
SOM 230-
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mTOR inhibitors may represent a possible medical therapy for
aggressive neuroendocrine tumors
everolimus reduces cell viability of
invasive neuroendocrine tumors in vitro
with possible cooperative effects with SOM230
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clinical trials are needed to confirm
these promising pre-clinical findings
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Phase 3 Placebo-Controlled Study of Everolimus in Patients Receiving Sandostatin LAR for Advanced Carcinoid Tumors
Phase 3 Placebo-Controlled Study of Everolimus in Advanced Pancreatic Neuroendocrine Tumors
Primary end-point: Response Rate; Recruitment closed: 160 patients
(PUBLISHED JCO)
Primary end-point: PFS; Recruitment closed: 415 patients
(PRESENTED AT THE ASCO GI 2011)
Primary end-point: PFS; Recruitment closed: 410 patients
(PUBLISHED NEJM)
Phase 2 Open Label Study of Everolimus in Advanced Pancreatic Neuroendocrine tumors after Failure of Chemotherapy
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Prolongation of progression-free survival in several NETS
efficacy and safety of daily everolimus for the treatment of advanced patients with nonfunctioning NETs, including bronchial carcinoid
everolimus slows tumor growth and improves progression free survival
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APPLICATIONS OF RAD001 IN
ENDOCRINE-RELATED TUMORS
Sezione di Endocrinologia
Dipartimento di Scienze Biomediche e Terapie Avanzate
Università degli Studi di Ferrara
Direttore Prof. Ettore degli Uberti
Biomolecular analysis
Pathology
SurgeryMTC
20 patients: 6 ♂and 14 ♀
age = 50 ± 3.8 yr
19 primary tumors
1 metastatic lymph node
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N° Sex AgePlasma CT (pg/ml) TNM Stage Inheritance
# 1 M 46 157 T2N0Mx II SP
# 2 M 40 1500 T2N0Mx II FMTC
# 3 F 35 97 T2N0Mx II SP
# 4 F 31 306 T1N0Mx II SP
# 5 M 47 940 T2N0Mx II MEN2A
# 6 M 39 1500 T2NxMx II SP
# 7 F 35 700 T2N0Mx II SP
# 8 F 32 28 T1N+M0 II MEN2A
# 9 F 35 74 T1N0Mx I SP
# 10 F 33 19 T1N0M0 I FMTC
# 11 F 42 153 T2N0Mx II SP
# 12 M 52 207 T2N0Mx II SP
# 13 F 44 2350 T2N0Mx II SP
# 14 M 56 1258 T2N0Mx II SP
# 15 F 79 1500 T2N+Mx III SP
# 16 F 71 2578 T4N+M+ III SP
# 17 F 73 3848 T1N+M1 IVc SP
# 18 F 75 1500 T3N0Mx III SP
# 19 F 69 3405 T2N+Mx III SP
# 20 F 70 9227 T4N1bM+ IVb SP
Patients
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In vitro experiments:cell viavility
(colorimetric method)
Primary coltures
Freezer –80°C
MCZ
20 MTC
1 µM RAD001 10 nM SOM2301 nM to 1 µM RAD001
50 nM IGF-1
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Human MTC primary cultures
20 samples
14 responders
6 non responders
Cell viability response to
RAD001
primary cultures
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Responders Non responders p
Age (yr) 40,5 ± 2,1 72,8 ± 1,7 <0,01
F/M 8:6 6:0 -
Plasma calcitonin (pg/ml)
663,5 ± 203,8 3676,3 ± 1289,8 <0,01
Stage I or II III or higher -
Patients
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0
20
40
60
80
100
120
140
CT 10 nM 100 nM 1 uM
% c
ell
viab
ilit
y vs
. con
trol
all
RAD responders
RAD non responders
****
RAD001 significantly reduces cell viability in responder MTC
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RAD001 abrogates the proliferative effect of IGF-I
0
20
40
60
80
100
120
140
CT 10 nM 100 nM 1 µM
% c
ell vi
abili
ty v
s. c
ontr
ol
w/o IGF-I
with IGF-I
** **
*
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RAD001 significantly reduces p70S6K phosphorylation and abrogates the stimulatory
effects of IGF-I
0
20
40
60
80
100
120
140
160
0 10 nM 100 nM 1 uM
p70
S6
K p
hos
phor
ylat
ion
(% v
s. c
ontr
ol)
w/o IGF-1
with IGF-1
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0
5
10
15
20
25
SSTR1 SSTR2 SSTR3 SSTR4 SSTR5
MT
C
All (20)
RAD responders (14)
RAD non responders (6)
MTC predominantly express SSTR1 and SSTR2
Responder MTC lack SSTR5 expression
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0
15
30
45
60
75
SS
TR
1
SS
TR
2
SS
TR
3
SS
TR
4
SS
TR
5
mR
NA
cop
ies
x 1
04/µ
g to
tal R
NA
All
RAD responders
RAD non responders
Responder MTC lack SSTR5 expression
MTC predominantly express SSTR1 and SSTR2
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SOM230 potentiates RAD001 antiproliferative effects
0
20
40
60
80
100
120
CT 10 nM 100 nM 1 uM
% c
ell vi
abili
ty v
s. c
ontr
ol
w/o SOM230
with SOM230
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Everolimus might represent a possible
therapeutic tool in MTC, also in association
with SRIF analogs
In conclusion
• RAD001 reduces cell viability in 70% of MTC primary cultures
• MTC responder coltures do not express SSTR5
• the antiproliferative effect of RAD001 is blocked by IGF-1 and enhanced by co-treatment with SOM230
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RAD001 in 7 GH-omas
RAD001 dose-dependently reduced somatotropinoma cell viability in vitro
*P<0.05
0
20
40
60
80
100
120
CT 1 nM 10 nM 100 nM 1 µM
% c
ell vi
abili
ty * *
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RAD001 in GH-omas
*P<0.05; **P<0.01
RAD001 is blocked by IGF-1
0
20
40
60
80
100
120
% c
ell vi
abili
ty
CT IGF1 50 nM100 nM 1 µM
RAD
100 nM 1 µM
RAD + IGF1 50 nM
* *
120
0
20
40
60
80
100
% c
ell vi
abili
ty
100 nM 1 µM
RAD
SOM 10 nM 100 nM 1 µM
RAD+ SOM 10 nM
CT
* * ** SOM230 enhances RAD001 effects
EFE 2012
Colture primarie
RAD001 in GH-omasGH secretion
0
20
40
60
80
100
120
140
Ct RAD001 SOM230 SOM+RAD IGF-1 RAD+IGF-1
% v
s. c
ontr
ol
* *
RAD001 reduces GH secretionIGF-1 co-treatment blocks this effect
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Colture primarie
Biomolecular analysis
Pathology
Pituitary Unit TNS surgery
10 ACTH-secreting pituitary adenomas
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Colture primarie
In vitro experiments:cell viavility (colorimetric method)apoptosis with caspase 3/7 assays mTOR phosphorylation by ELISA
Primary coltures
Freezer –80°C
MCZ
10 nM –1 µM RAD001
50 nM IGF-1
10 nM SOM230
EFE 2012
Colture primarie
*P<0.05; **P<0.01
0
20
40
60
80
100
120
0 10 nM 100 nM 1 µM
Everolimus
Cel
l vi
abili
ty (
% v
s. c
ontr
ol)
***
0
20
40
60
80
100
120
140
0 10 nM 100 nM 1 µM
Everolimus
casp
ase
3/7
act
ivit
y (%
vs.
con
trol
)
** **
RAD001 dose-dependently reduces cell viability, induces apoptosis and inhibits p70S6K phosphorylation
0
20
40
60
80
100
120
0 10 nM 100 nM 1 µM
Everolimus
p70
S6
K p
hos
phor
ylat
ion
(% v
s. c
ontr
ol)
****
EFE 2012
Colture primarie
RAD001 blocks the effects of IGF-I on cell viability, apoptosis and p70S6K phosphorylation
cell
viab
ility
(%
vs.
con
trol
)
0
20
40
6080
100
120
140
160
0 100 nM 1 µM
Everolimus
w/o IGF-I
with IGF-I
casp
ase
3/7
act
ivit
y (%
vs.
con
trol
)
0
20
40
60
80
100
120
140
0 100 nM 1 µM
Everolimus
w/o IGF-I
with IGF-I
p79
S6
K a
ctiv
ity
(%vs
. co
ntro
l)
0
20
40
60
80
100
120
140
160
0 100 nM 1 µM
Everolimus
w/o IGF-I
with IGF-I
**
**
***
** **
**
****
*P<0.05; **P<0.01
EFE 2012
Colture primarie
cell
viab
ility
(%
vs.
con
trol
)
0
20
40
60
80
100
120
0 100 nM 1 uM
Everolimus
w/o SOM230
with SOM230**
**
**
SOM230 enhances the antiproliferative effects of RAD001
*P<0.05; **P<0.01
EFE 2012
Colture primarie
• RAD001 dose-dependently inhibits ACTH-secreting
pituitary adenoma cell viability in primary culture by
inducing apoptosis and inhibiting p70S6K activity
• These effects are blocked by IGF-I
• SOM230 enhances RAD001 effects
CONCLUSION
everolimus might represent a possible
medical treatment aiming at
controlling ACTH-secreting adenomas
These preliminary results indicate
that everolimus might represent a
possible medical treatment aiming at
controlling pituitary adenoma growth
CONCLUSION
EFE 2012
Colture primarie
•Evaluate antisecretory activity on GEP NETs
•Evaluate the possible influence of RAD001 on
chemoresistance
•Evaluate the possible influence of mTOR
inhibition of GH peripheral effects
FUTURE PERSPECTIVES
EFE 2012
Colture primarie
EFE 2012
Colture primarie
1: Molè D, Gentilin E, Gagliano T, Tagliati F, Bondanelli M, Pelizzo MR, Rossi M, Filieri C, Pansini G, Degli Uberti EC, Zatelli MC. Protein kinase C: a putative new target for the control of human medullary thyroid carcinoma cell proliferation in vitro. Endocrinology. 2012 May;153(5):2088-98.
2: Gagliano T, Filieri C, Minoia M, Buratto M, Tagliati F, Ambrosio MR, Lapparelli M, Zoli M, Frank G, Degli Uberti E, Zatelli MC. Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion. Pituitary. 2012 Feb 21. [Epub ahead of print] PubMed PMID: 22350942.
3: Lee M, Theodoropoulou M, Graw J, Roncaroli F, Zatelli MC, Pellegata NS. Levels of p27 sensitize to dual PI3K/mTOR inhibition. Mol Cancer Ther. 2011 Aug;10(8):1450-9..
4: Molè D, Gagliano T, Gentilin E, Tagliati F, Pasquali C, Ambrosio MR, Pansini G, Degli Uberti EC, Zatelli MC. Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors. Endocr Relat Cancer. 2011 Jul 1;18(4):439-50.
5: Martínez-Fuentes AJ, Molina M, Vázquez-Martínez R, Gahete MD, Jiménez-Reina L, Moreno-Fernández J, Benito-López P, Quintero A, de la Riva A, Diéguez C, Soto A, Leal-Cerro A, Resmini E, Webb SM, Zatelli MC, degli Uberti EC, Malagón MM, Luque RM, Castaño JP. Expression of functional KISS1 and KISS1R system is altered in human pituitary adenomas: evidence for apoptotic action of kisspeptin-10. Eur J Endocrinol. 2011 Mar;164(3):355-62.
6: Zatelli MC, Tagliati F, Amodio V, Buratto M, Pelizzo M, Pansini G, Bondanelli M, Ambrosio MR, Degli Uberti EC. Role of pituitary tumour transforming gene 1 in medullary thyroid carcinoma. Anal Cell Pathol (Amst). 2010;33(5):207-16.
EFE 2012
Colture primarie
7: Zatelli MC, Minoia M, Martini C, Tagliati F, Ambrosio MR, Schiavon M, Buratto M, Calabrese F, Gentilin E, Cavallesco G, Berdondini L, Rea F, degli Uberti EC. Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids. Endocr Relat Cancer. 2010 Jul 28;17(3):719-29.
8: Zatelli MC, Minoia M, Filieri C, Tagliati F, Buratto M, Ambrosio MR, Lapparelli M, Scanarini M, Degli Uberti EC. Effect of everolimus on cell viability in nonfunctioning pituitary adenomas. J Clin Endocrinol Metab. 2010 Feb;95(2):968-76
9: Zatelli MC, Ambrosio MR, Bondanelli M, Uberti EC. Control of pituitary adenoma cell proliferation by somatostatin analogs, dopamine agonists and novel chimeric compounds. Eur J Endocrinol. 2007 Apr;156 Suppl 1:S29-35. Review. Erratum in: Eur J Endocrinol. 2007 Oct;157(4):543.
10: Zatelli MC, Piccin D, Vignali C, Tagliati F, Ambrosio MR, Bondanelli M, Cimino V, Bianchi A, Schmid HA, Scanarini M, Pontecorvi A, De Marinis L, Maira G, degli Uberti EC. Pasireotide, a multiple somatostatin receptor subtypes ligand, reduces cell viability in non-functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion. Endocr Relat Cancer. 2007 Mar;14(1):91-102.
11: Zatelli MC, Piccin D, Tagliati F, Bottoni A, Luchin A, Vignali C, Margutti A, Bondanelli M, Pansini GC, Pelizzo MR, Culler MD, Degli Uberti EC. Selective activation of somatostatin receptor subtypes differentially modulates secretion and viability in human medullary thyroid carcinoma primary cultures: potential clinical perspectives. J Clin Endocrinol Metab. 2006 Jun;91(6):2218-24.
12: Zatelli MC, Piccin D, Tagliati F, Bottoni A, Ambrosio MR, Margutti A, Scanarini M, Bondanelli M, Culler MD, degli Uberti EC. Dopamine receptor subtype 2 and somatostatin receptor subtype 5 expression influences somatostatin analogs effects on human somatotroph pituitary adenomas in vitro. J Mol Endocrinol. 2005 Oct;35(2):333-41.
EFE 2012
Colture primarie
13: Zatelli MC, Luchin A, Piccin D, Tagliati F, Bottoni A, Vignali C, Bondanelli M, degli Uberti EC. Cyclooxygenase-2 inhibitors reverse chemoresistance phenotype in medullary thyroid carcinoma by a permeability glycoprotein-mediated mechanism. J Clin Endocrinol Metab. 2005 Oct;90(10):5754-60.
14: Zatelli MC, Maffei P, Piccin D, Martini C, Rea F, Rubello D, Margutti A, Culler MD, Sicolo N, degli Uberti EC. Somatostatin analogs in vitro effects in a growth hormone-releasing hormone-secreting bronchial carcinoid. J Clin Endocrinol Metab. 2005 Apr;90(4):2104-9.
15: Zatelli MC, Piccin D, Bottoni A, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Taylor JE, Culler MD, Cavazzini L, degli Uberti EC. Evidence for differential effects of selective somatostatin receptor subtype agonists on alpha-subunit and chromogranin a secretion and on cell viability in human nonfunctioning pituitary adenomas in vitro. J Clin Endocrinol Metab. 2004 Oct;89(10):5181-8.
16: Zatelli MC, Piccin D, Bondanelli M, Tagliati F, De Carlo E, Culler MD, Uberti EC. An in vivo OctreoScan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro. Horm Metab Res. 2003 Jun;35(6):349-54.
17: Zatelli MC, Piccin D, Tagliati F, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Culler MD, degli Uberti EC. Somatostatin receptor subtype 1 selective activation in human growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas: effects on cell viability, GH, and PRL secretion. J Clin Endocrinol Metab. 2003 Jun;88(6):2797-802.
EFE 2012
Colture primarie
Maria Chiara ZatelliSezione di Endocrinologia
Dipartimento di Scienze Biomediche e Terapie AvanzateUniversità degli Studi di Ferrara
Via Savonarola 944121 Ferrara
Tel: 0532 455859 – 237272Fax: 0532 236514
E-mail: [email protected]